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I:\MANUALS\MANUALS\Karnal Bunt\2004KB\Append D.fm

D

Karnal Bunt

Appendix D 1

Laboratory Procedures

Contents

Introduction and Background D-1

Grain Analysis Methods D-3

Background D-3

Obtaining Samples D-3

Automated Methods D-3

High Speed Optical Sorting Method D-3

Grain Inspection Machine Method D-6

Quality Control Procedures D-11

Manual Method D-12

Identifying Tilletia indica D-14

Storing Positive Kernels D-16

Decontaminating Labware and Instruments D-17

Seed Analysis Methods D-18

Background D-18

Setting Up the Laboratory D-18

Receiving Samples D-19

Extracting Samples D-21

Preparing Slides D-33

Examining Slides for Karnal Bunt D-38

Training Technicians and Verifiers D-41

Selecting and Training Bunted Kernel Verifiers D-43

Preparing New Sieves for Use D-45

Maintaining Sieves D-46

Ordering Sieves D-47

Forms D-47

Introduction and Background

The purpose of the laboratory section is to describe procedures for 

examining wheat grain samples for the presence of Tilletia indica

infected grain (i.e., bunted kernels) and seed samples for the presence 

of T. indica teliospores. Grain analysis laboratories normally process 

grain samples from fields, bins, or national surveys for the presence of 

bunted kernels. Seed laboratories process seed samples and perform 

extraction procedures to determine the presence of T. indica 

teliospores.
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Introduction and Background

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In program areas where bunted kernel analysis precedes seed analysis 

for spores, seed lots from regulated areas must be negative for bunted 

kernels prior to being considered for spore testing, and seed samples 

may also be processed at grain labs for the detection of bunted 

kernels. However, because of contamination potential, a different 

subsample of that seed lot must be submitted for spore analysis than 

the one processed for bunted kernel analysis and that subsample 

should not enter grain analysis laboratories unless it is sealed in 

nalgene bottles and is unopened. Some program areas analyze seed 

samples first for spores, then do a bunted kernel analysis on positive 

spore lots.

Grain and seed analysis laboratories must be physically separate and 

personnel working in grain laboratories (or field personnel) should not 

enter a seed analysis laboratory without proper decontamination 

safeguards.

The details of these procedures may be modified to individual 

laboratory needs; however, basic analysis methods and contamination 

safeguards should not be deviated from. Some seed analysis 

laboratories may wish to do the spore wash first and then the analysis 

of bunted kernels from the remaining dry sample afterward, and that 

is permissible.

Be very careful to maintain the identity of all grain and seed samples, 

with careful, accurate labeling.

The techniques for extracting teliospores by selective sieving were 

developed by Gary Peterson and Morris Bonde1, United States 

Department of Agriculture, Agricultural Research Service (USDA, 

ARS), Ft. Detrick, MD. Plant Protection and Quarantine (PPQ) Plant 

Pathologist Ted Boratynski developed the grain inspection machine for 

bunted kernel analysis. Arizona Department of Agriculture Plant 

Pathologist Ron Ykema and others at the State Agriculture Laboratory 

in Phoenix, Arizona assisted in refinement of all the techniques 

described here and in the preparation of this manual. Joel Floyd, as 

PPQ Area Identifier in Nogales, AZ, wrote the teliospore and bunted 

kernel identification sections.

1 Peterson, G. L. and M. R. Bonde. 2000. Size-selective sieving for detecting teliospores of Tilletia indica

in wheat seed samples. Plant Disease 9:999-1007.
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Grain Analysis Methods

Laboratories for bunted kernel detection should be in an enclosed area 

and equipped with the supplies listed below. A process for logging in 

samples received should be in place. The area designated for grain 

analysis must be physically separate from the seed (spore) analysis 

laboratory.

Background

This subsection describes three methods used to identify wheat 

kernels infected with Tilletia indica, the causal agent of Karnal bunt. 

These infected kernels are called bunted kernels. Included are specific 

procedures for decontamination of samples and facilities.

Obtaining Samples

The laboratory manager will specify which types and the priority of 

samples to be examined for bunted kernels. If assigned the task of 

detecting bunted kernels, do not enter any seed analysis areas.

Automated Methods

The automated methods are preferred for analyzing grain samples for 

bunted kernel detection. There are two types of automated methods in 

use. One involves optical high speed sorting technology and the other 

employs the use of a grain inspection machine. The optical scanner, 

where available, is used for both samples from the regulated area and 

National Survey samples. When grain inspection machines or high 

speed optical sorting machines are not available, the manual method 

can be used.

High Speed Optical Sorting Method

Supplies Laboratory personnel will need the following supplies to examine 

wheat kernels for Karnal bunt using (Satake®) optical sorting 

technology:

◆ Bags, paper

◆ Bags, plastic

◆ Beaker, glass or plastic, 600-mL

◆ Bleach (6%), 1 gallon

◆ Bottle, spray

◆ Boxes, slide holder

Important

Seed samples tested for bunted kernels and/or spores must not utilize the 

same subsamples.
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◆ Brush, fine tip applicator

◆ Brushes, paint

◆ Cover slips, glass

◆ Dust masks

◆ Fluorescent paint/dye

◆ Forceps

◆ Gloves, latex

◆ Illuminators, microscope

◆ Labels for slides

◆ Lab towels, disposable

◆ Ladder

◆ Lamp, magnifying

◆ Markers, permanent, fine-point, black

◆ Microscopes, compound

◆ Microscopes, dissecting

◆ Nail polish, clear

◆ Needle probes

◆ Paper towels

◆ Parafilm

◆ Petri dish, plastic disposable

◆ Razor blades

◆ Reinforcements, white

◆ Shear’s mounting medium (see Preparing Shear’s Mounting 

Medium for preparation)

◆ Slides, glass

◆ Slides, identification aid

◆ Tape, packing

◆ Tape, quarantine

◆ Tissue, toilet

◆ Trash can, small

◆ Tub, small

◆ Tubs, 18-gallon/sample storage

◆ Ultra-violet light

◆ Vacuum cleaner, large shop type

◆ Vacuum cleaner, small portable
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Procedure The protocol for the high speed optical sorting method (using the 

Satake® Scanmaster II) follows:

At the beginning of the day and about every four hours thereafter:

Step 1—

Make sure the machine is on for at least 30 minutes before use and 

that the valve for the compressor hose is at the “ON” position.

Step 2—

Check the light levels. Both should be at about 12.0–14.4 V (Setup 1 

Menu).

Step 3—

Select the MEDIUM Preset (Setup 3 menu). Be sure to press RECALL 

after selecting the correct product. When you go back to the Main 

menu, make sure the proper Preset is showing at the top of the screen.

Step 4—

Spike the sample with 3-13 fluorescent QC kernels. Recovery of spiked 

kernels will be done using the manual seed inspection machine.

Step 5—

Weigh the rejects. Based on a 4-lb sample, rejects will run between 

180g–320g, depending on the color of the wheat and the number of 

darkened kernels.

Step 6—

Adjust the DARK TRIP as needed to achieve the appropriate 

percentage of rejects (10-15% depending on variety). Light colored 

samples require higher trip rates. Red winter wheat trip rates are 

between 784 and 796. Durum wheat trips are between 844 and 864.

Step 7—

Open and clean the machine. Check the feeder, chute, and sorting 

chamber for kernels.

For each sample:

Step 1—

Clean the sample as needed.

Step 2—

Select the correct pre-set based on sample color and variety.

Before opening front panel ensure that the ejectors are turned off. Failure to 

do so could result in damage to the ejectors!
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Step 3—

Run the sample.

Step 4—

Open and clean the machine. Check the feeder, chute, and sorting 

chamber for remaining kernels.

Repeat Steps 1 through 4 for each sample.

To shut down the sorter:

Step 1—

Press the “X” in the upper right hand corner of the screen. This should 

get you back to the computer “desktop.”

Step 2—

Press “START” in the lower left hand corner.

Step 3—

Press “SHUTDOWN.”

Step 4—

A window will appear that says “What do you want the computer to 

do?” The “SHUTDOWN” command should already be selected. Press 

“OK.”

Step 5—

When the screen says “It is OK to turn off your computer,” press the 

power switch on the upper left side of the sorter. Do NOT shut down 

the sorter without going through this process.

Step 6—

Turn off the air supply valve.

Grain Inspection Machine Method

Supplies Laboratory personnel will need the following supplies to examine 

wheat kernels for Karnal bunt using the grain inspection machine 

method:

◆ Beakers, glass, 600-mL

◆ Bleach, 6 percent solution

◆ Cover slips, glass (any size)

◆ Dazzlens, glass cleaner

◆ Dish, petri, plastic disposable, 50 x 9 mm

◆ Dust mask or respirator
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◆ Forceps, specimen, stainless steel, large tip, small tip

◆ Gloves, latex, small, medium, and large

◆ Grain inspection machine

◆ Kimwipes XL

◆ Labels, white paper

◆ Microscope, compound

◆ Microscope, dissecting

◆ Nail polish, clear, toluene/formaldehyde free

◆ Paint brushes

◆ Plastic bags, 12 x 12 inch, zip-lock

◆ Shear’s mounting medium (see Preparing Shear’s Mounting 

Medium for preparation)

◆ Slides, microscope, 3” x 1”

◆ Tape, labeling

◆ Tech Spray, inert dusting gas, or vacuum with hose attachment

◆ Vials, plastic

◆ Worksheets, Karnal bunt kernel analysis, in-house

Preparing the 

Workstation

Designated bunted kernel readers must wear clothing that will prevent 

sample contamination and protect them from harmful chemicals.

Step 1—Put on protective clothing.

Put on a dust mask or respirator, and latex gloves as needed. If the 

sample shows pink or violet dye, wear a dust mask when examining 

the grain. The dye shows that the grain has been treated with 

pesticide.

Step 2—Prepare the grain inspection machine.

Turn the feeder rheostat control knob on the grain inspection machine 

counter clockwise to the OFF position (see Figure D-1 for a diagram of 

a grain inspection machine). Turn ON the power switch located on the 

control panel. Place a clean petri dish and a pair of clean forceps on 

the work surface to the right side of the feeder trough. Open the 

acrylic lid above the grain hopper.

Analyzing the 

Sample

Step 1—Pour the sample into the hopper.

Obtain the sample to be tested and record the Karnal bunt sample 

number of the worksheet. Carefully open the sample bag(s) without 

tearing. Being careful not to over-fill, pour a portion of the sample into 

the grain hopper and close the acrylic lid.
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Grain Analysis Methods

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Step 2—Activate the vibrating mechanism.

Turn ON the light switch. Turn ON the feeder rheostat control knob 

(vibrator switch). Adjust the grain hopper discharge opening and the 

feeder rheostat control until you get the desired feed rate. Only one 

level of grain should cover the bottom of the feeder trough as it 

vibrates, permitting observation of the kernels from all sides.

Important

Be careful to empty the grain bin cup frequently to avoid spillage. You may wish 

to pour only a cup full of sample at a time into the grain hopper.
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FIGURE D-1: Grain Inspection Machine
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Step 3—Check for bunted kernels.

When you observe a suspect bunted kernel, turn down the feed rate or 

the rheostat control and use forceps to move the kernel to the petri 

dish. Take the suspect bunted kernels to the dissecting microscope 

and examine for the presence of teliospores. Place all of the bunted 

kernels into a clean plastic vial or in a sealed petri dish and prepare a 

paper label to include in the vial with the Karnal bunt number, your 

initials, and the date. Using a black marker label the outside of the 

vial/petri dish. Follow identification authority verifier’s training 

procedures. If teliospores are present, prepare a microscope slide 

using Shear’s mounting medium. Seal the cover slip with nail polish. 

Review the slide using the compound microscope, verifying the 

characteristics of the T. indica teliospore. Have an identifier with 

identification authority verify the presence of T. indica on the kernels 

found in a sample. Decontaminate the forceps with bleach and dispose 

of or disinfect the used petri dish.

Step 4—Record results.

Record your results on the appropriate worksheet. For positive results, 

record the total number of bunted kernels found in the sample. Place 

all positive samples in the storage area for positive samples. Place all 

negative samples in the storage area for negative samples, or dispose 

of as per project guidelines.

Step 5—Clean the inspection machine.

Clean the grain inspection machine as follows: Turn OFF the power 

switch and the light switch. Remove the seed bin cup and tilt the unit 

to permit any loose kernels to fall out through the seed bin cup area. 

Use inert dusting gas to blow any kernels from the unit. Return the 

unit to its upright position. Clean the magnifying lens with Dazzlens 

and Kimwipes. Periodically open the top of the machine and use paint 

brushes, or a vacuum with a hose attachment, or high pressure air to 

clean kernel pieces and dust from the inside of the machine.

Step 6—Clean the area and remove protective clothing.

When all of the samples have been examined, sweep up any spilled 

grain and dispose of it in a disposal bag in the area. Remove all 

protective clothing and place in the disposal bag.
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Quality Control Procedures

The following are quality control procedures for detection of Tilletia 

indica bunted kernels.

As part of Quality Control (QC) procedures, samples are spiked with 

QC kernels, Tilletia indica bunted kernels, to measure efficacy of the 

Satake Optical Sorter and grain inspection machine personnel. These 

QC kernels are actual bunted kernels painted with a dye that 

fluoresces under UV light. Collected data will include the number of 

QC kernels added to the sample and the number of recovered QC 

kernels. The laboratory supervisor will use this data to keep the 

project director informed concerning validity of the screening 

procedures, and to provide feedback to lab personnel.

◆ Each lab will have a vial containing 16 bunted kernels, painted 

with fluorescent paint, with which to “spike” the QC sample.

◆ The 4-lb QC sample consists of previously tested negative grain 

and 3-13 QC kernels determined by the random number 

generator.

Facilities 

Equipped with 

Satake Optical 

Sorter 

Technology

Step 1—

Use the random number generator to determine the number of bunted 

kernels with which to spike the QC sample.

Step 2—

Place the bunted kernels into the QC sample and run through the 

sorter at the lowest acceptable Dark Trip (784).

Step 3—

Look at the QC sample “discard container” with the UV light to 

ascertain that all of the QC kernels were ejected.

◆ If all of the QC kernels are found, proceed to next step.

◆ If any QC kernels are missing, look at clean portion container 

with the UV light to determine if the sorter missed any QC 

kernels. If it is determined that QC kernels were missed, raise 

the Dark Trip one unit (i.e. 784 to 788) and return the QC 

kernels to the QC sample and run again. Repeat this process 

until all QC kernels are ejected into the discard portion 

container. This will be the Dark Trip value to use for one shift 

(every four hours). Run a QC sample for each shift.

Step 4—

Replace all QC Kernels into discard portion. Have each lab technician 

working in the lab look at the discard using a manual inspection 

station (bunt machine) until he or she finds one bunted kernel.
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Step 5—

Remove and secure all bunted kernels from the QC sample into a safe 

container and safeguarded in an appropriate location.

◆ Appropriate worksheets will be provided for the daily recording of 

all QC results (see Figure D-3).

Field Facilities 

without Satake 

Optical Sorter 

Technology

Step 1—

The laboratory supervisor should spike each 4-lb QC sample with 3-13 

fluorescent QC kernels. Determine the number of kernels per sample 

by using a random number generator.

Step 2—

Inspect the sample using the manual grain inspection machine until 

all the QC kernels are found.

Step 3—

Record quality control information (see Figure D-3). If all QC kernels 

are recovered, no further action is required. If all QC kernels are not 

recovered, screen the 4-lb sample using the portable UV light to insure 

the indicated number of QC kernels were present.

Manual Method

Laboratory personnel will need the following supplies items to examine 

wheat kernels for Karnal bunt using the manual method:

Supplies ◆ Bags, disposal, 12" x 24"

◆ Beaker, glass, 600-mL

◆ Bleach, 6 percent solution

◆ Bottle, spray, 1-L, hardware store

◆ Boxes, cardboard

◆ Cover slips, glass (any size)

◆ Dish, petri, plastic disposable, 50 x 9 mm

◆ Dish pans, plastic, department store

◆ Forceps, specimen, stainless steel, large tip

◆ Gloves, latex, small, medium and large

◆ Labels, white paper

◆ Lamp, magnifying

◆ Microscopes, dissecting and compound

◆ Nail polish, clear, toluene/formaldehyde free

◆ Shear’s mounting medium (see Preparing Shear’s Mounting 

Medium for preparation)
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◆ Slides, microscope, 3" x 1"

◆ Storage units

◆ Towels, lab

◆ Vials, plastic

◆ Wash basin, double sided

◆ Worksheets, Karnal Bunt Kernel Analysis, in-house

Preparing the 

Workstation

Designated bunted kernel readers must wear clothing that will prevent 

sample contamination and protect them from harmful chemicals.

Step 1—Put on protective articles.

Put on a dust mask or respirator, and latex gloves as needed. If the 

grain samples show pink or violet dye, wear a dust mask when 

examining the grain. The dye indicates that the grain has been treated 

with pesticide.

Step 2—Set up items.

Take one 600-mL beaker, one petri dish, one large forceps, and one 

plastic tray. Set these items up at your workstation and use the 

appropriate worksheet for recording data.

Analyzing the 

Sample

Step 1—Scoop grain into the beaker.

From the sealable plastic bag holding the grain sample, scoop 

approximately 500 mL of grain and pour it into the beaker. Take the 

sample to your workstation for examination.

Step 2—Spread the sample.

Spread a portion of the sample throughout the plastic tray in 

approximately one layer of grain. Do a visual overview of the sample in 

the tray, looking for grain that shows a darkening on one end or 

obvious deterioration of the grain tissue.
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Step 3—Check for bunted kernels.

When you observe a suspect bunted kernel, use forceps to move the 

kernel to the petri dish. Take the suspect bunted kernels to the 

dissecting microscope and examine for the presence of teliospores. 

Place all of the bunted kernels into a clean plastic vial or a sealed petri 

dish and prepare a paper label to include in the vial with the Karnal 

bunt number, your initials, and the date. Using a black marker label 

the outside of the vial. Follow identification authority verifier’s training 

procedures. If teliospores are present, prepare a microscope slide 

using Shear’s mounting medium. Seal the coverslip with nail polish. 

Review the slide using the compound microscope, verifying the 

characteristics of the T. indica teliospore. Have an identifier with 

identification authority verify the presence of T. indica on the kernels 

found in a sample. Decontaminate the forceps with bleach and dispose 

of or disinfect the used petri dish.

Step 4—Record results.

Record your results on the appropriate worksheet. For positive results, 

record the total number of bunted kernels found in the sample. Place 

all positive samples in the storage area for positive samples. Place all 

negative samples in the storage area for negative samples, or dispose 

of as per project guidelines.

Step 5—Clean the work area.

Clean and disinfect the work area with bleach solution.

Identifying Tilletia indica

Early stages of infection first appear at the germ end of the wheat 

kernel. These symptoms are usually not visible when the grooved side 

is facing down. Therefore, an effective technique for the manual 

method is to turn many seeds over to look for symptoms.

The first visible stage of Karnal bunt infection is a swollen area that 

appears gray or dark at the germ end forming a triangular shaped area 

on either side of the groove (see illustrations A2, B1, and B2 on the 

laminated insert titled Symptoms of Karnal Bunt (Tilletia indica) 

and Black Point). This is the thin pericarp, or outer epidermal layer, 

of the seed coat through which a darkening is visible and is evidence 

of a mass of teliospores beneath (the sorus). This swollen area of the 

pericarp may already be ruptured or can easily be punctured with a 

teasing needle, exposing many dark brown teliospores.
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0

FIGURE D-2: Symptoms of Karnal Bunt (Tilletia indica) and Black Point
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Karnal bunt teliospores, large by fungal spore standards, are easy to 

see with a hand lens. However, to verify Karnal bunt symptoms from a 

seed sample, viewing the infected kernels under a dissecting 

microscope will be necessary to see if teliospores are present. They 

appear as hundreds or thousands of small, granulate, shiny dark 

brown to black spheres, resembling microscopic ball bearings or 

buckshot. They are contained within the area of the seed that the 

fungus has consumed, together forming the sorus.

Refer to the laminated insert. More advanced infection (A3 and A4) in 

the wheat kernel manifests itself as a blackening along the groove, 

progressively consuming more of the inner portions of seed tissue. A 

very advanced stage of infection results in the wheat kernel taking on 

the appearance of a boat or dugout canoe (A5).

Black point is a disease of non-quarantine importance caused by a 

variety of fungal species (Alternaria spp. and Helminthosporium spp.). 

The superficial symptoms of Karnal bunt have been confused with 

those of black point, but close examination reveals distinct 

differences. While both diseases give the germ end of the wheat a 

darkened appearance, black point is actually more of a darkening or 

discoloration of the pericarp (C1 and C2, laminated insert). Upon 

examination under a dissecting microscope, no spores, nor anything 

that resembles teliospores, are visible. Also, the darkening caused by 

black point organisms may extend farther from the tip of the seed and 

be more general in its occurrence on the seed, often appearing on the 

ventral and dorsal surfaces. Black point shows no obvious erosion of 

the seed tissue as in Karnal bunt.

Other smut diseases such as common bunt (T. laevis or T. tritici) may 

occur in the samples. Consult with a bunted kernel identifier to verify.

To make a positive identification for any spore, making a 

microscope slide will be necessary to view the spores under high 

magnification.

Storing Positive Kernels

Step 1—Place bunted kernels in a plastic vial or petri dish.

Place all of the bunted kernels from the same sample in a plastic vial 

or petri dish. Using a black marker, label the vial with the sample 

number, your initials, and the date on the outside of the vial, and 

inside the vial on a white label.
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Step 2—Verify presence of Tilletia indica.

Have an identifier with identification authority for the bunted kernel 

verify the presence of T. indica on the kernels found in a sample. 

Return all negative grain to its original packaging. Also return the 

positive sample (minus any bunted kernels) to its original packaging 

and place it in the positive storage area. Verify the presence of T. indica 

teliospores on the kernel by making a slide and storing all positive 

slides with proper labels. Retain voucher slides for a project-specified 

time in a secure storage container.

Step 3—Record findings.

Record your findings, initials, and date on the appropriate analysis 

worksheet (Figure D-3). Have the verifier initial in the appropriate 

space for the positive samples verified.

Step 4—Take care of used equipment.

Place all labware and instruments into the dirty bin for 

decontamination. Use new gloves, labware, and lab tools for each new 

sample. Continue with the next sample.

Decontaminating Labware and Instruments

Follow this procedure to decontaminate labware and instruments 

used in manual or automated methods:

Take all dirty labware and lab tools to a designated area for 

decontamination. As a precaution, keep labware “wet” until 

decontamination with the bleach solution. Soak all labware and lab 

tools in a 30 percent bleach solution for 15 minutes, rinse with clear 

water, dry, and return to the bunt area for the next use.

After examining all the samples, or at the end of a shift, 

decontaminate the area. Decontamination includes these activities:

◆ Bleach all workstation surfaces (hoods, chairs, tables, etc.).

◆ Make sure no dirty labware or instruments remain in the area.

◆ Sweep the floor and mop with 30 percent bleach solution.

When decontamination is complete, dispose of all shoe covers, hair 

covers, coveralls, and gloves into the biohazard container. Dispose of 

all waste during the examination process into the biohazard container. 

Turn off all hoods and lights when finished.

Important

Handle all samples as if positive to help prevent contamination.
Appendix D Laboratory Procedures

Seed Analysis Methods

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Seed Analysis Methods

Background

The purpose of seed analysis laboratories is to determine the presence 

or absence of Karnal bunt, T. indica, teliospores in submitted wheat 

seed samples. For a seed lot to be considered negative, five replications 

must be performed and all five tests must be negative for the presence 

of T. indica teliospores. Seed lot samples with only spore fragments 

that cannot be measured can be resampled and run a second time.

Setting Up the Laboratory

Whether laboratory personnel process a few samples per day or a 

hundred, the same minimum facility requirements are necessary. By 

meeting these requirements, each laboratory will have the flexibility to 

use its available resources, while still maintaining a national 

standard. The seed analysis laboratory must have three basic areas:

◆ A reception area

◆ A dirty room

◆ A clean room

Combining functions performed in the dirty room and the clean room 

into one room is acceptable under the following conditions:

◆ Samples for processing come from a non-Karnal bunt area.

◆ No positive samples have been in the laboratory.

Once a suspect positive sample is encountered, these rooms and 

functions need to be separate. Also, workers should always process a 

negative control wheat sample with each group of samples once any 

samples entering the laboratory test Karnal bunt positive.

Reception Area The reception area, a place for initial delivery of samples and 

decontamination of packaging, must be outside the laboratory 

facilities, whether literally outside or in a designated building, loading 

dock, or shed. This area can also be used for storing the remaining 

sample not used in the laboratory evaluation. The area must have 

access to running water.

Important

Field sample personnel or individuals entering the grain analysis laboratory are 

not allowed entry into the seed analysis laboratory without proper 

decontamination safeguards. Showering and changing clothes is usually 

sufficient.
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Dirty Room The dirty room is an area for opening dry seed samples, adding 

extraction solution, and transferring or weighing samples. The 

primary concern is to prevent dry, loose teliospores from becoming 

airborne and contaminating other samples and the external surfaces 

of the extraction equipment. The minimum equipment required is a 

clean air station or biological containment hood, to prevent direct air 

movement and ventilation from this room into the clean room. 

Between each sample, workers must wipe down all working surfaces 

with detergent and 30 percent bleach (6 percent sodium hypochlorite). 

Technicians must change gloves between samples and not wear 

contaminated garments into clean areas.

Clean Room No sample should enter the clean room unless it is in liquid 

suspension. Workers moving from the dirty room into the clean room 

should have separate lab coats. Activities performed in the clean room 

are sample extraction, slide preparation, extraction ware 

decontamination, and microscopy. Laboratory personnel must 

decontaminate the extraction table surfaces between sets of samples. 

Workers must clean up any spills before they dry and spray the area of 

any spill with 30 percent bleach. After extracting samples on trays, 

remove and decontaminate the trays.

Receiving Samples

Background This subsection describes procedures used to receive seed samples at 

approved laboratories for the analysis for Tilletia indica. Included are 

specific procedures for decontaminating the seed samples before they 

enter the laboratory, and log-in procedures.

Supplies Laboratory personnel will need the following supplies to receive the 

samples:

◆ Bleach, (sodium hypochlorite), 6 percent

◆ Cardboard boxes

◆ Centrifuge tubes

◆ Chain-of-custody seals (if appropriate)

◆ Container to hold bleach solution

◆ Coveralls

◆ Dish pans, plastic, department store

◆ Garden hose with nozzle, hardware store

◆ Karnal bunt spore analysis worksheets, in-house designed

◆ Lab coats

◆ Latex gloves

◆ Plastic disposal bags, 12" x 24"
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◆ Safety glasses

◆ Secure storage units for samples

◆ Spray bottle, 1-liter, hardware store

◆ Utility cart

Sample 

Decontamination

When samples arrive at the laboratory, make sure to receive them at a 

designated check-in/decontamination area. This area must be 

physically separate from the laboratory facility where samples are 

processed. A wash basin and hose setup is necessary at this location. 

Use the following procedure to check in and decontaminate the 

samples before bringing them inside the laboratory. Wear gloves, 

safety glasses, and a lab coat or coveralls during this procedure.

Step 1—Check samples.

Check each sample against the accompanying paper work to ensure 

matching sample number. Check all seals and samples for integrity.

Step 2—Decontaminate samples.

Decontaminate the sample containers using Table D-1 as a guide:

TABLE D-1: Decontamination of Samples

Step 3—Log-in samples.

Take the paperwork and samples, if necessary, into the sample 

receiving area of the laboratory for log-in. 

If the samples (or 

subsamples) are in: Then:

Plastic bottles 1. REMOVE the bottles from the sample bag.

2. TIGHTEN the lids to ensure a water tight seal.

3. SUBMERGE each bottle in a 30 percent bleach solution 

prepared fresh daily.

4. After 15 minutes, RINSE the bottles with tap water from a 

garden hose.

5. GO to Step 3.

Plastic bags 1. MAKE sure the bags are sealed tightly.

2. FILL a spray bottle with 30 percent bleach solution, prepared 

fresh daily.

3. SPRAY the bags with the bleach solution and ALLOW the 

solution to remain on the bags for 15 minutes.

4. After 15 minutes, RINSE the bags with tap water from a garden 

hose.

5. GO to Step 3.

Important

Never take any samples into the laboratory before decontaminating them.
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Sample Log-In Accurate sample log-in is very important. Be very careful to maintain 

each sample’s identity.

Step 1—List the sample number on the worksheet.

List the assigned sample number on the analysis worksheet, and label 

sample containers with the assigned laboratory number. Place 

samples for spore analysis on a utility cart after logging-in and 

labeling them. Enter a “Quality Assurance (QA) Negative” laboratory 

control on the worksheet as well. Make sure to include on the cart a 

label for the control (for assembly in the weighing room). Use positive 

laboratory controls occasionally to prove the laboratory’s ability to 

detect and identify positive samples.

Step 2—Label the centrifuge tubes.

Label centrifuge tubes with laboratory sample numbers for each 

sample in each set. Place the tubes in a disposable tube rack supplied 

with the tubes, and label the rack with the set number. Place the 

samples, worksheet, and centrifuge tubes on a utility cart and deliver 

these items to the weighing area or the solution addition area.

Reserve Sample 

Storage

Using approved sample journals, label excess sample bags and bottles 

with correct laboratory sample numbers. Sign and date any broken 

seals. Reseal the samples and dispose of used gloves in a biohazard 

bag at the decontamination area. Group samples by box number and 

place them in cardboard boxes in a secure storage area. Identify the 

boxes appropriately.

Extracting Samples

Background This section describes procedures used at approved laboratories to 

extract teliospores of Tilletia indica from wheat or other crop samples. 

Included are specific procedures for preparation of extraction solution 

and preparation of Shear’s mounting medium. Also included are 

procedures used to prepare extraction containers and measure out 

samples, and an alternative method for adding extraction solution to 

premeasured bottled samples. This section also details the extraction 

technique itself.

Supplies Laboratory personnel will need the following supplies to process the 

samples:

◆ Aprons, rubber, 45" x 35"

◆ Bags, biohazard, 38" x 48"

◆ Bags, biohazard, 12" x 24"

◆ Balance, analytical

◆ Beakers, glass, 2-L and 1-L
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◆ Beakers, 600 mL

◆ Bleach (6 percent sodium hypochlorite)

◆ Bottle, glass, 1-L with ground glass stopper

◆ Bottle, spray, plastic, 1-L, hardware store

◆ Bottles, HDPE wide mouth, 250-mL, plastic

◆ Bottles, wash, 500-mL

◆ Box, broken glass disposal

◆ Bulb, pipette

◆ Carboy, 20-L with spigot

◆ Cart, glassware

◆ Cart, utility, stainless steel

◆ Centrifuge, minimum 1,000 r/min required

◆ Citric acid monohydrate (C6H8O7.1H2O)

◆ Cups, disposable, paper, 5-oz

◆ Cups, disposable, paper, 3-oz

◆ Cups, portion, paper, 1-oz

◆ Cylinders, graduated, 25-mL, 250-mL, 500-mL, 1-L

◆ Deionized water

◆ Detergent

◆ Dissecting microscope, linen tester, or 10X magnifying glass

◆ Dust masks

◆ Ethyl alcohol, 95 percent, anhydrous

◆ Flasks, Erlenmeyer, graduated, 500-mL

◆ Funnels, glass, 60-mm ID top x 13-mm OD stem

◆ Funnels, glass, 100-mm ID top, 18-mm OD stem

◆ Glasses, safety

◆ Gloves, gauntlet, sizes 9, 10, and 11

◆ Gloves, latex, surgical

◆ Gloves, nitrile, sizes 8, 9, 10, and 11

◆ Glycerol

◆ Hood, biosafety with HEPA filter

◆ Hoods, fume

◆ Inverted light microscope, equipped with ocular micrometer
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◆ Isopropyl alcohol, 70 percent

◆ Karnal Bunt Sieve Log

◆ Karnal Bunt Sieve Usage Log

◆ Labels, 1" x 2", 3 up x 10, Avery, 5160

◆ Liner, bench (substitute for plastic trays)

◆ Magnifying glass, illuminated

◆ Mixer, vortex, thermolyne

◆ Parafilm

◆ Pipette tips, 200-1000 μL, for Eppendorf adjustable pipettor

◆ Pipette tips, repeating

◆ Pipettes, Mohr, 1-mL and 10-mL

◆ Pipettor, adjustable, Eppendorf, 100-1000 μL

◆ Pitcher, 3000-mL

◆ Potassium acetate (KC2H3O2)

◆ Rack, test tube

◆ Scoop, lab, stainless steel

◆ Shaker, orbital, thermolyne

◆ Sieve, nylon, 20 μm pore with square openings, 3" ID x 50 mm 

high, USDA2

◆ Sieve, nylon, 53 μm pore with square openings, 3" ID x 50 mm 

high, USDA2

◆ Silicone sealant

◆ Sodium phosphate, dibasic, anhydrous (Na2HPO4)

◆ Stage micrometer

◆ Stir bars, magnetic, 11⁄2" x 1⁄2"

◆ Stirrer, magnetic

◆ Syringes, 10 ml

◆ Tape, lab, blue, 1"

◆ Tape, lab, red, 1⁄2"

◆ Tape, yellow stretchy, lab safety

◆ Terri-wipes

◆ Timers

2 See attachment for construction of the sieves.
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◆ Trays, plastic, 18" x 12" x 1⁄2" deep (substitute for bench liners)

◆ Tubes, centrifuge, disposable, plastic, conical, 15-mL

◆ Tubs, disinfectant

◆ Tubs, self-draining and holding

◆ Tween 20 or wetting agent

Preparing the 

Extraction 

Solution

Prepare the extraction solution in a separate, clean room. Never bring 

grain samples into this room.

Step 1—Fill a carboy with deionized water.

Wearing safety glasses and latex gloves, fill a 20-L carboy with 18 L of 

deionized water.

Step 2—Add detergent.

Using a graduated cylinder, add 12 mL Tween 20 detergent or wetting 

agent to the carboy. Rinse the cylinder several times with deionized 

water into the carboy.

Step 3—Label the carboy.

Place a strip of red lab tape at the 18-L mark and label as Tween 20 

solution. Cap tightly and roll the carboy on a utility cart to make sure 

that the Tween is well dispersed in the water.

Preparing 

Shear’s 

Mounting 

Medium

After centrifuging aqueous extracts of the sample and decanting the 

supernatant, re-suspending the remaining pellet in mounting fluid is 

necessary. Shear’s mounting medium, the standard mounting 

medium for smut fungi, provides excellent buffering capacity. The 

fluid maintains the structural integrity of the Karnal bunt spores, 

while limiting evaporation. Additionally, the glycerol in the Shear’s 

fluid maintains a refractive index of 1.47 at 27°C, which allows the 

effective transmission of light by the compound microscope. Shear’s 

mounting fluid preserves sample extracts for future microscopic 

evaluation. You can use the procedure for preparation below, or 

purchase the mounting fluid from a commercial source.

Preparing 1 L of 0.2 M Na2HPO4 solution

Step 1—Weigh anhydrous Na2HPO4.

Wearing safety glasses and latex gloves, weigh 38.39 g of anhydrous 

Na2HPO4 into a tared 2-L beaker on a 3 decimal balance.

Step 2—Add deionized water.

Using a graduated cylinder, add 1 L of deionized water to the beaker 

and stir with a magnetic stirring bar until all granules are dissolved.
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Step 3—Transfer the solution.

Using a funnel, transfer the solution to a 1-L glass stoppered bottle 

and label as 0.2 M Na2HPO4 with the date of preparation and your 

initials.

Preparing 1 L of 0.1 M citric acid solution

Step 1—Weigh citric acid monohydrate.

Weigh 21.01 g of citric acid monohydrate into a tared 2-L beaker on a 

3 decimal balance.

Step 2—Add deionized water.

Using a graduated cylinder, add 1 L of deionized water to the beaker 

and stir until all granules are dissolved.

Step 3—Transfer the solution.

Using a funnel, transfer the solution to a 1-L glass stoppered bottle 

and label as 0.1 M citric acid solution with the date of preparation and 

your initials.

Preparing 600 mL of Shear’s Mounting Medium

Step 1—Transfer Na2HPO4.

Using a pipette, transfer 19.45 mL of 0.2 M Na2HPO4 into a 1-L beaker.

Step 2—Transfer citric acid.

Using a 1-mL pipette, transfer 0.55 mL of 0.1 M citric acid solution 

into the same beaker.

Step 3—Add potassium acetate and deionized water.

Add 6 g of potassium acetate and 280 mL of deionized water to the 

beaker.

Step 4—Stir to dissolve solids.

Stir with a magnetic stirring bar until all solids are dissolved and the 

solution is uniform in appearance.

Step 5—Add glycerol and ethyl alcohol.

Continue stirring and add 120 mL of glycerol and 180 mL of 95% ethyl 

alcohol to the beaker.

Step 6—Transfer the solution.

After the solution is uniform in appearance, use a funnel to transfer to 

a 1-L glass stoppered bottle. Label as Shear’s mounting medium, and 

include the date of preparation and your initials.

Step 7—Clean glassware.

Clean all glassware with lab detergent and rinse with tap water followed 

by deionized water. Place pipettes in the pipette washer with tips up.
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Optional Method 

for Preparing 

Extraction 

Flasks

Prepare and label an Erlenmeyer flask containing extraction solution 

for each sample that requires weighing or measuring out before 

extraction. Carry out the following procedure in the room designated 

for preparing extraction solution:

Step 1—Fill flasks with extraction solution.

Wearing safety glasses and latex gloves, fill 500-mL flasks to the 100 

mL mark with extraction solution directly from the 20-L carboy. Place 

two 3-oz disposable paper cups over the mouth of each flask 

immediately after adding the solution.

Step 2—Label flasks.

Label one flask for each sample in the set to be extracted. Place the 

flasks for that set on the top shelf of a utility cart in the order listed on 

the worksheet and move it to the sample weighing area.

Step 3—Place worksheet and tubes on cart.

Place the spore analysis worksheet and labeled centrifuge tubes on the 

top shelf of the cart. Place the samples in order from left to right on the 

second shelf of the cart and take the cart to the room designated for 

measuring out the samples.

Weighing and 

Measuring the 

Samples

It is preferable to have 50-g pre-measured samples arrive at the 

laboratory in sealed nalgene bottles that are disinfected with chlorine 

solution before entering the laboratory. Use these procedures when 

larger, unmeasured, or dirty samples arrive at the seed testing 

laboratory.

Take samples that require weighing to the sample weighing area. It is 

recommended that this procedure be carried out in a biosafety hood 

fitted with a HEPA filter. Generally, the only samples of this type are 

bin sweepings or other “dirty” samples that contain mostly dirt and 

chaff and very little grain. Measure most other samples by volume 

rather than by weight. Consult your supervisor if weighing or 

measuring unusual sample matrices or sample types is necessary. 

Wear safety glasses, latex gloves, and a lab coat or overalls.

Sanitizing the hood

Before weighing or measuring any samples, first sanitize the hood 

thoroughly.

Step 1—Turn on power.

Turn on power to the hood, light, and balance.

Step 2—Sanitize the hood.

Thoroughly sanitize the interior of the hood (including walls, glass 

door, and ceiling) by spraying with 30 percent bleach solution. This 

also includes the balance and the area under the balance. Let the 

bleach stand for 15 minutes.
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Step 3—Spray hood again.

Spray the hood again with the bleach solution and wipe dry with terry 

wipes. Dispose of used terry wipes in the biohazard bag.

Step 4—Clean bleach residues from hood.

Spray the entire hood with 70 percent isopropyl alcohol and wipe dry. 

Doing this will help to clean bleach residues from hood surfaces, 

particularly the glass window of the hood. Dispose of used terry wipes 

in the biohazard bag.

Weighing the samples

For samples that are very dirty and contain very little sample by 

weight, measure them out by weighing at least a 2.0 g portion of 

sample, up to 50 g, on an analytical balance. Wear a dust mask when 

handling dirty and dusty samples.

Step 1—Take a sample from the cart.

From the cart containing the sample set to be weighed, take a sample 

and make sure that the sample number and the number on the 

container of extraction solution are identical. Place the sample and the 

corresponding container in the hood. Pull down the hood door to allow 

only enough space to move sample containers in and out of the hood.

Step 2—Put on gloves.

Put on a new pair of latex gloves; place one of the two paper cups on 

the balance and tare it.

Step 3—Mix the sample.

Mix the sample well by inverting and rolling thoroughly. Open the 

sample container and use one paper cup to scoop out 2.0 g ± 0.1 g of 

sample into the cup on the balance. Transfer excess sample that has 

not touched the hood or balance back to the sample container.

Step 4—Transfer the contents.

Transfer the contents of the paper cup to the sample container and 

seal the opening with parafilm.

Step 5—Close the sample container.

Close the sample container and place the cups in the palm of one 

gloved hand and fold that glove over them. Now place this glove in the 

palm of the other hand and repeat. Dispose of the used materials in 

the biohazard bag.

Step 6—Remove the sample from the hood.

Remove the sample and the container from the hood and place the 

container back on the cart. Return the sample to the storage area.
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Step 7—Repeat procedure for additional samples.

For additional samples, repeat Steps 1 through 6 using new gloves for 

each sample. Deliver the cart to the extraction lab after weighing all 

samples. Return sample bags to the storage area.

Step 8—Maintain cleanliness.

Always maintain cleanliness of the weighing area. If any sample falls 

on the balance or the hood floor, do not place in any sample container. 

Dispose of the sample with the gloves and cups at the end of the 

weighing cycle. If any sample falls on the hood or balance, sanitize the 

area of the spill before weighing the next sample. Wipe up the spilled 

sample and dispose of it in a biohazard bag. Spray the area where the 

sample fell and the surrounding area with the bleach solution. Wipe 

up the bleach solution and dispose of the dirty gloves and terry wipes 

in the biohazard bag.

Step 9—Resanitize the hood and balance.

Resanitize the hood and balance after each sample.

Measuring the samples

Measuring the samples by volume is preferable to measuring by 

weight. Measure by volume these types of samples:

◆ Samples that do not arrive premeasured in plastic bottles

◆ Samples that require additional analyses

Measure these samples into clean glass or new plastic sample 

containers.

Step 1—Measure the samples.

The procedure is the same as for weighing samples in the previous 

section, except that this procedure involves measuring the samples by 

scooping three 1-oz cupfuls into a container (may be different for other 

types of crops). Use the paper portion cups for this purpose and then 

dispose of them in the biohazard bag.

Step 2—Take plastic bottles by cart.

When measuring the samples into plastic bottles, take them by cart to 

add extraction solution before taking them to the extraction lab. See 

Adding Extraction Solution below.

Step 3—Measure control wheat.

Also, measure control wheat into containers using the above 

procedure. Seal these containers of control wheat and store them in 

the solution addition room for later use in extraction sets as needed.
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Step 4—Resanitize the hood.

When finished weighing or measuring samples for the day or at the 

end of a shift, resanitize the hood.

Adding 

Extraction 

Solution

When an extraction set consists of premeasured samples in plastic 

bottles, add the extraction solution to the bottles in the hood in the 

solution addition room. Take only decontaminated samples into this 

room. Wear safety glasses and latex gloves throughout this procedure.

Step 1—Turn on power and sanitize the hood.

Turn on power to the hood and sanitize the hood as described in the 

subsection Sanitizing the hood. Pull down the hood door to allow only 

enough space to move sample bottles in and out of the hood.

Step 2—Check samples.

Before adding solution to any of the bottles, first check to make sure 

that each sample on the utility cart has a matching sample number 

listed on the worksheet. The bottles should also appear to contain 

approximately 50 g of sample for wheat; other crops may be different. 

If any bottle appears to contain too much or too little sample, call it to 

the attention of your supervisor before measuring a new sample from 

the bag of grain retained in storage.

Step 3—Add extraction solution.

For each sample, place bottle(s) in the hood and put on a new pair of 

latex gloves. Add extraction solution from the carboy on the counter 

outside the hood to a clean, disposable paper cup. Fill to a level equal 

to the mark on the reference cup next to the carboy. This should be 

approximately 100 mL of solution. Place the cup of solution in the 

hood and open the sample bottle. Add the solution to the bottled 

sample. Tighten the bottle cap and place the sample back on the cart. 

After completing a sample, place the cup(s) in the palm of one gloved 

hand and fold that glove over it. Now place this glove in the palm of the 

other hand and repeat. Dispose of the gloves and cup in the biohazard 

bag. Repeat this procedure for each sample on the cart, using a new 

pair of gloves and cup for each sample. Do not reuse paper cups. Once 

extraction fluid has been added to the sample containers, samples 

must be extracted within 30 minutes.

Step 4—Resanitize the hood.

When finished weighing or measuring samples for the day or at the 

end of a shift, resanitize the hood.

Extracting the 

Samples

The clean room must have sufficient counter space (approximately 15 

ft) to carry out the extractions. The lab must also have a shaker table, 

a chemical fume hood for bleach baths, and sinks to rinse glassware. 

A separate counter top is necessary to support the centrifuge station.
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Extract the samples in a clean room, always wearing safety glasses. As 

an added precaution, wear protective clothing such as a rubber apron 

and heavy rubber gloves when decontaminating glassware with bleach 

solution. Wear latex gloves when extracting samples and change the 

gloves between samples.

Setting up for extraction

Step 1—Position bench liner or plastic trays.

Place the laboratory bench liner on the bench top with the plastic side 

down. To prevent confusion when processing multiple samples, use 

lab tape to mark out lanes approximately 30 cm wide. Another 

approved method is to use individual plastic trays (18" x 12" x 1⁄2") for 

each sample. Plastic trays will contain spills, and tubes cannot roll 

into the wrong lane. The trays also work well at smaller labs that do 

not have room to set up multiple lanes.

Step 2—Lay out beaker and sieve sets.

Place one 600-mL beaker toward the rear of each lane and set a 20 μm 

pore nylon sieve on top of it. Place another 600-mL beaker in front of 

the first beaker and set a 53-μm pore nylon sieve on top of that 

beaker. Lay out as many beaker and sieve sets as there are samples to 

extract, and place centrifuge racks between each set. Place small glass 

funnels (60 mm ID) in the center of the bench, along with pitchers and 

wash bottles containing tap water. When using the plastic tray option, 

keep all the extraction equipment for one sample in that sample’s tray. 

Assure that flasks, beakers, and centrifuge tubes are properly labeled 

with corresponding sample numbers.

Step 3—Prepare bleach baths.

Prepare bleach baths by filling large disinfectant tubs inside fume 

hoods (or in a separate room) with 30 percent bleach solution. The 

bleach solution baths need to contain a sufficient volume of solution 

to completely submerge the glassware being decontaminated.

Extracting the samples

Step 1—Shake samples.

Place sample bottles or flasks containing extraction solution on an 

orbital shaker. Secure the containers in the shaker so that the fit is 

appropriate for the type of container used. Shake samples for 5 

minutes at 200 r/min. Do not allow samples to remain in the 

extraction solution for longer than 30 minutes due to release of starch 

granules. While samples are shaking, remove the centrifuge tubes that 

arrived with the samples from the disposable rack and place one per 

lane on the bench top. Place the disposable centrifuge tube rack and 

worksheet in the center of the bench. Sanitize the utility cart with 30 

percent bleach solution before returning it to the sample receiving 

area.
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Step 2—Place samples with tubes.

After shaking the samples, place each in the lane/tray with the tube 

that corresponds to that sample. Double check to make absolutely 

sure that tubes and sample bottles have matching sample numbers.

Step 3—Pour sample into 53-μm sieve3.

For each sample, invert the bottle or swirl the flask to re-suspend the 

grain and debris. Immediately pour the contents into the 53-μm sieve, 

collecting the filtrate in the 600-mL beaker. Add an additional 100 mL 

of water to the container, swirl or recap and shake the bottle, and then 

pour into the sieve to rinse the grain. Take care to avoid splashing the 

sample solution during sieving procedures. Reextract samples if 

cross-contamination occurs. Rinse a minimum of twice, preferably 

three times, tilting the sieve on top of the beaker to aid draining. Place 

the empty extraction container on the glassware cart.

Step 4—Pour filtrate into 20-μm sieve3.

After the sieve has drained, discard the grain into a small disposal 

bag, and place the used sieve on a glassware cart. Pour the filtrate 

from the beaker into the 20-μm sieve, collecting the filtrate in the 

second 600-mL beaker. Add an additional 100 mL of water to the first 

beaker, swirl, and pour through the sieve. Use a wash bottle to rinse 

down the side walls of the beaker, and pour this rinsing into the sieve 

as well. Rinse a minimum of twice, preferably three times. Place the 

used beaker on the glassware cart.

Step 5—Rinse the nylon screen.

Once the 20-μm sieve has drained, tilt the sieve on top of the beaker 

and use the wash bottle to rinse down the sides and upper portion of 

the nylon screen. Tip the sieve over the 50-mL beaker at a 45° angle 

and use a wash bottle with a steady stream to rinse debris into the 

beaker. Repeat this rinse twice. Debris will pool in the lower portion of 

the sieve.

3 Follow quality assurance checks for sieves.

The sieves are manufactured with a ring to make them stackable as an option 

for using them together on one beaker.
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Step 6—Rinse debris into the centrifuge tube.

Open the centrifuge tube and place it upright in the centrifuge tube 

rack. Use a wash bottle to rinse debris from the 20-μm sieve into the 

tube. You may also use a small glass funnel to facilitate this transfer. 

To assure that all tubes have the same amount of liquid, fill the tubes 

with liquid to just below the screw cap threads. Recap the tube and 

return to the disposable centrifuge rack. Pour the extraction filtrate 

into a 20-L carboy containing 1⁄2 gallon of bleach. Place filtrate from 

samples treated with fungicide in a separate carboy. Place used 

glassware and sieve on the glassware cart. As an added precaution 

against contamination by airborne spores, soak all used labware in a 

water bath when not in use, until decontamination.

Step 7—Repeat procedure for each sample.

Repeat Steps 3 through 6 for each sample in the extraction set. If 

extracting multiple samples becomes necessary, processing all 

samples through one step before proceeding to the next step is most 

efficient. Be sure to remain undistracted while doing this. Take 

care not to mix samples or confuse the extraction steps.

Step 8—Notify your supervisor in case of spills.

In case of a sample spill, notify your supervisor to decide if a second 

extraction of the sample is necessary. If so, discard the sample and 

show the result for that sample as “L” on the worksheet with your 

initials and date. Record all lab accidents in the lab notebook kept at 

the centrifuge station. If a sample spills on the bench liner, replace it 

with a clean liner. If any sample contacts the bench surface, spray a 

30 percent bleach solution on the area to decontaminate it, and wipe 

with a clean towel.

Step 9—Transfer glassware and sieves to bleach bath.

Immediately transfer all used glassware and sieves from the glassware 

cart to a bleach bath and soak them for 15 minutes. Do not allow 

sieves to remain in the bleach bath for more than 15 minutes, as this 

will rapidly degrade the nylon screens. Rinse glassware and sieves 

thoroughly with hot tap water. Hang glassware on a rack to drain and 

place sieves in self-draining plastic tubs. Discard plastic sample 

bottles after decontaminating them in the bleach bath.

Step 10—Centrifuge the tubes.

After preparing all of the tubes for the set, take them to the centrifuge 

station and centrifuge at 1,000 r/min for 5 minutes. Pour off the 

supernatant and re-suspend the pellet by pipetting 500 μl of Shear’s 

mounting medium into the tube. Recap and vortex a few seconds to 

mix the pellet with the Shear’s fluid. Record the set information in the 

centrifuge log book before delivering the centrifuge tubes and 

worksheet to the microscope area.
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Cleaning Up the 

Extraction Lab

Clean up the clean room by following these steps:

Step 1—Remove the bench liner.

At the end of each extraction series or as necessary, remove the bench 

liner and discard in a large biohazard bag along with the small bags of 

extracted grain and other potentially contaminated materials. Tie 

these bags shut with lab tape. Place in red biohazard containers in a 

designated hazardous waste area. When using plastic trays, removing 

and discarding bench liners is not necessary.

Step 2—Dispose of the filtrate.

Pour the bleached sample filtrate from the carboy down a sink drain 

as necessary. After filling any carboys containing filtrate from seed 

treated with pesticide, pour the contents into a labeled hazardous 

waste container.

Step 3—Sanitize surfaces.

Spray all bench tops, carts, and other potentially contaminated 

surfaces with 30 percent bleach solution. Wipe all these surfaces 

down at the end of each extraction series. Sweep and mop the floor in 

the clean room with bleach water at the end of each extraction series 

as well.

Step 4—Sanitize pitchers and wash bottles.

Soak all pitchers and wash bottles in 30 percent bleach solution for at 

least 15 minutes at the end of each shift. Take care to rinse these 

items thoroughly before using them again.

Preparing Slides

Background This section describes procedures used to prepare sample slides for 

microscopic examination for Tilletia indica teliospores. Included are 

specifications for lab garments and set-up of the working area. The 

section also identifies priorities in the processing of samples. Topics 

include receiving sample tubes, preparing slides, filling out slide labels 

and data sheets, preparing quality assurance slides, handling spills, 

and cleaning up. Keep the slide preparation area separate from other 

sample processing areas.

Supplies Laboratory personnel will need the following supplies to prepare the 

slides:

◆ Applicators, cotton-tipped

◆ Bags, biohazard 38" x 48"

Fumes from chlorine bleach solutions can be hazardous if inhaled. Make sure 

the area is properly ventilated and consider having personnel use a respirator 

during disinfection.
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◆ Bags, biohazard 12" x 24"

◆ Beakers, 150-mL, 250-mL, and 400-mL

◆ Bleach (6 percent sodium hypochlorite)

◆ Bottles, plastic, narrow mouth

◆ Bottles, plastic, spray

◆ Box, microscope slide

◆ Boxes, glass disposal, floor

◆ Bulbs, rubber pipette

◆ Cabinet, storage

◆ Caps, bouffant, 21"

◆ Centrifuge, minimum 1,000 r/min required

◆ Cleanser, skin, antimicrobial

◆ Coats, lab, polyethylene, medium, large, and extra large

◆ Cover slips, microscope, 22 x 50 mm

◆ Cytoseal 60, low viscosity

◆ Flasks, Erlenmeyer, 500-mL and 1,000-mL

◆ Fume absorber

◆ Gloves, latex, medium and large

◆ Graduate, transfer pipettes, polyethylene, 5-mL

◆ Labels, multi purpose, white

◆ Petroleum jelly (100 percent)

◆ Pipettes, Pasteur, disposable, 9-inch

◆ Pipettes, transfer, Padl-Pet 0.5-mL

◆ Scissors

◆ Shear’s mounting medium

◆ Slide warmer

◆ Slides, microscope, 3" x 1"

◆ Teri Reinforced Towels

◆ Tray, slide, aluminum or plastic

Store all supplies in a designated Karnal bunt cabinet. Designate a 

clean area specifically for slide preparation. Clean all surfaces in this 

area with a 30 percent bleach solution before setting up the work area.
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Setting-Up the 

Working Area

Prepare the slides under a fume extractor or canopied laminar flow 

hood to minimize exposure to chemicals. Keep a supply of Shear’s 

solution on hand. During the process of slide making, Shear’s solution 

is necessary to dilute dense samples or to add liquid under the cover 

slip as needed.

Step 1—Disinfect surfaces.

Before making any slides, disinfect surfaces with 30 percent bleach 

solution from a spray bottle.

Step 2—Fill plastic bottles.

Fill separate plastic bottles with Cytoseal and Shear’s solution. Label 

the bottles.

Step 3—Fill a container with bleach.

Fill a container half full with 30 percent bleach solution for disposal of 

Pasteur pipettes.

Step 4—Add bleach to a plastic bottle.

Label a large plastic screw-cap bottle with “Quarantine Material.” Add 

200 mL of 30 percent bleach. Place the bottle by the centrifuge. Use 

this bottle to decant supernatant from centrifuged samples.

Step 5—Use new supplies for each sample.

Use new slides, cover slips, and pipettes for each sample. Check 

supplies and restock if needed. Place plastic pipettes, Pasteur pipettes, 

and padl-pets in separate, clean beakers.

Step 6—Turn on the slide warmer.

Under a hood place one layer of reinforced towel. On top of the towel, 

place a colored sheet of paper (throw away between samples). Turn on 

the slide warmer to approximately 58°C.

Identifying 

Sample Priority

Sample processing priority will vary by region. Laboratory managers 

will establish sample priorities based on program needs. Contact the 

laboratory manager if sample priority is in question.

Processing 

Sample Tubes

Step 1—Count tubes and data entries.

Count the tubes and data entries on the sheet. Make sure that the 

sample number matches the data sheet number, along with the set 

number. Report discrepancies to the laboratory manager.

Preparing the 

Slides

Step 1—Extract the pellet.

Use a new piece of paper, pipettes, gloves, slide(s) and cover slip(s) for 

each sample. Use clean pipette bulbs for each sample if not using 

cotton-plugged Pasteur pipettes. Move the sample tube to be prepared 

to the front of the rack. Put on new gloves. Use a Pasteur pipette with 

a pipette bulb to extract the whole pellet at the bottom of the tube. 
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Squeeze the pipette bulb before inserting into the tube. Slowly release 

the pressure on the pipette bulb to extract the pellet. Avoid extracting 

excess supernatant to reduce the number of slides required per 

sample pellet. However, transferring the entire pellet to slides is 

important.

Step 2—Homogenize dense samples.

When processing dense, muddy, or thick samples, introduce the 

Pasteur pipette into the sample tube and homogenize the sample by 

pressing the pipette bulb several times. Homogenize the sample 

between each slide. Maintain pressure on the pipette bulb and remove 

the pipette from the tube. Hold the pipette at approximately a 45° 

angle above the slide and expel two or three small drops onto the slide 

depending on the sample density. For dense samples, dispense two 

drops of sample onto the slide and using a plastic pipette, place a drop 

of Shear’s mounting medium in the middle. Doing this will help 

produce a nonlayered, uniform slide.

Step 3—Spread the sample.

Use the end of the Pasteur pipette to spread the sample to a small 

rectangle. Avoid placing excess sample on the slide to eliminate 

seepage, or preparing a slide that will be too dense to read easily. Use 

more slides if necessary.

Step 4—Place a cover slip on the slide.

Return the tube with Pasteur pipette to the sample rack or pipette 

bucket. Place a cover slip over the drops on the slide. Do this by 

holding the cover slip above the slide at approximately a 45° angle and 

then touching the drop nearest the edge of the slide with the edge of the 

cover slip. A padl-pet pipette may help to move the opposite side of the 

cover slip down slowly until it touches the slide. This may help to 

reduce air under the cover slip. Remove air bubbles only by tapping 

lightly on the cover slip. Do not lift the cover slip to remove air bubbles.

Step 5—Position the cover slip.

Leave a 1⁄4 inch margin between the cover slip and the slide edge 

opposite the label. Place the Pasteur pipette in 30 percent bleach 

solution. Cap the sample tube and discard the tube only after making 

the label.

Step 6—Seal the cover slip.

Seal the slides if you are unable to read them immediately. All positive 

voucher slides must be sealed for storage.
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Dispose of the padl-pet pipette in a disposal bag. Seal the cover slip to 

the slide after preparing all of the slide(s) from a sample. Using a 

plastic pipette, extract the needed amount of Cytoseal. Seal the long 

sides of the cover slip, then the narrow sides to the slide. Release the 

Cytoseal (or clear nail polish) slowly, running the pipette along the 

edge of the cover slip. Avoid making a pass over an area more than 

once, but inspect the perimeter of the cover slip for missed or poorly 

sealed areas. Touch up with Cytoseal as needed. Discard excess 

Cytoseal from the plastic pipette into a plastic screw cap bottle for 

disposal. Keep containers of Cytoseal and Shear’s mounting medium 

closed when not in use. Remove gloves and discard along with sample 

tubes, colored paper, and plastic pipettes into the biohazard bag.

Making Slide 

Labels and 

Filling Out the 

Data Sheet

Make slide labels only for the current sample. Never use correction 

fluid on slide labels; make a new label.

Step 1—Prepare slide labels.

Prepare the label from top to bottom using a permanent ink pen as 

instructed in Table D-2.

TABLE D-2: Preparation of Slide Labels

Step 2—Label the slides.

Affix the label(s) to the slide(s). Record the number of slide(s) prepared 

for the sample in the comment section of the data sheet.

Step 3—Warm the slides.

Keeping the slide horizontal, place it on the warmer for 5 to 15 

minutes. Transfer slides to a slide tray. Repeat the slide preparation 

for each sample in the rack.

Step 4—Complete data sheet.

After completing a data sheet of samples, count the number of slides 

prepared and compare it with the total listed on the data sheet. Initial 

“Slide Preparer” section on the data sheet.

Description Slide Label (Example)

Sample Code CG-96-172

Number of slides prepared 1/3, 2/3, 3/3, etc.

Set Number Set 3052

Person who read the slide Reader Initials:

Authorized person who verified the positive finding of 

Tilletia indica or person who completed negative reread

Ver. Initials:

Date the sample was read Date:
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Step 5—Place tray and data sheet on counter.

Place the tray and data sheet on the counter marked for reading. After 

readers read the slides and record the results on the data sheet, they 

should return each slide to its appropriate sequential location in the 

slide tray.

Handling Spills Cover all spills of supernatant or sample with 30 percent bleach 

solution and a reinforced towel. Let soak for 15 minutes. Wipe up the 

bleach and rinse with water. Discard towels in a biohazard bag.

Cleaning Up Discard all used disposables, clean slide-making area with 30 percent 

bleach solution, and replace supplies. Turn off the extraction hood 

and slide warmer.

Examining Slides for Karnal Bunt

Background This section describes procedures used for the microscopic 

examination for and identification of teliospores that may be Tilletia 

indica. A compound microscope is necessary to examine the slides. 

Included are procedures for selecting, examining, and identifying 

Karnal bunt. The section also includes procedures for handling 

positive slides, negative slides, and record keeping. Microscopic 

identification requires a room with sufficient counter top and chair 

space to hold multiple microscope stations.

Selecting a Slide Select a slide based on the priority established by the laboratory 

manager. Before taking a slide, place your initials in the “Slide Taken 

By” box and record the slide number in the “Slide” box of the Tilletia 

indica Spore Analysis Worksheet (Figure D-6).

Examining the 

Slide

Examine the slide using the 10X objective and 10X ocular. Begin at 

one corner of the cover slip, including the area on the edge of the cover 

slip containing slide sealant. Scan the entire slide, either vertically or 

horizontally, left to right or right to left. Be consistent in your scanning 

procedure and be aware that the image is reversed. Take care to 

remember your direction if interrupted during a scan. A simple 

method is to place a pen or pencil on the table pointed in the direction 

of the scan at the time of interruption.

Identifying 

Karnal Bunt 

Teliospores

After carefully examining the Tilletia indica reference slides, and 

recognizing the variability in mature and immature spores, look for 

similar objects. Refer also to the laminated color illustration titled 

Karnal Bunt Teliospores, Tilletia indica Mitra. Upon finding a 

suspect spore, change the objective to 40X and look for the following 

characteristics:
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Mature Teliospores

◆ Spherical shape with an average diameter of 39 μm and a range 

of 24–47 μm. Atypical spores that are broken and/or irregularly 

shaped may be present.

◆ Color light brown to near black

◆ Smooth clear or yellowish sheath enveloping the spore that is 

visible when focusing on cross section of the spore

◆ Distinctive truncate projections that are visible on the 

circumference of the spore when focusing through a cross 

section and dark brown spots in the middle of the spore when 

focusing on the surface of the spore

Immature Teliospores Combined with Mature Teliospores

◆ Similar shape but often smaller than mature teliospores

◆ Color yellow to lighter brown

◆ More distinct, finer ornamentation than mature teliospores

Any of the above characteristics will suggest a suspect find of Tilletia 

indica teliospores.

Verifying 

Teliospores

Refer suspect teliospores (intact, broken, or fragmented) to the 

appropriate authority for verification.

If identified as T. indica, the verifier will do the following:

 1. Circle the position of the verified teliospore using a permanent 

marker.

 2. Print his or her initials on the slide label and in the “Verified By” 

column of the Tilletia indica Spore Analysis Worksheet.

If the teliospores are indicative of T. indica, but are undersized or 

comprised of fragments less than 50 percent, the verifier will follow the 

steps above and in addition will do the following:

 3. Record an asterisk (*) in the “# of Spores” column.

 4. Add the statement “undersized spores” or “fragments” to the 

“Comments” column on the Tilletia indica Spore Analysis 

Worksheet (Figure D-6).

Counting 

Teliospores as 

Positive

A seed sample will be considered positive for T. indica if at least one 

spore is found equal to or exceeding 33 μ in diameter and possessing 

morphological characteristics consistent with T. indica and not other 

Tilletia species (i.e., T. walkeri, T. horrida syn. T. barclayana). All 

teliospores used to make a positive determination for a sample must 

be verified by an identifier with identification authority for the T. indica

teliospore in absence of the host.
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Keeping 

Records

Maintaining accurate records of samples is extremely important. 

Always record information in ink. NEVER use correction fluid on 

laboratory documents, slide labels, or sample labels. If a correction is 

necessary, draw a line through the incorrect entry, enter the correct 

entry, and place your initials and date beside the correct entry.

Tilletia indica Microscopy Log Sheet

Keep your own record of the samples and their counts using the 

Tilletia indica Microscopy Log Sheet (Figure D-7). Record the sample 

#, set #, and 1st/2nd reads. Use the Sample # section to record slide 

number, if there are multiple slides. For example, “1 of 2 slides.” 

Record spore coordinates in the Comments section.

Slide labels

After completing a slide, place your initials and the date on the slide 

label.

Tilletia indica Spore Analysis Worksheet

Return the slide to the appropriate slide holder. Record the following 

data on the Tilletia indica Spore Analysis Worksheet (Figure D-6):

◆ Your initials

◆ The date

Have the verifier who confirmed the first T. indica teliospore record his 

or her initials under the Confirmed By section.

Interpreting 

Laboratory 

Results

A laboratory result, whether positive or negative, stands on its own. 

Repeating a test does not necessarily invalidate or validate the 

previous test unless the results are the same.

Rating on the Karnal Bunt Spore Analysis Worksheet

After a slide reader reads the slides and documents the results on the 

worksheet, the laboratory manager will rate the results using the 

following codes (only one rating is necessary per sample):

◆ P for positive. Used when at least one spore of T. indica is 

confirmed for the sample.

◆ N for negative. Used when no T. indica spores are confirmed for 

the sample.

◆ X for retest. Used when the verifier cannot make a positive 

identification.

◆ L for laboratory accident. Used when a sample is spilled, 

mislabeled, etc. Retesting is required.

◆ R for redundant. Used when there are multiple test results for 

the same sample, if a rating of P, X, or L is used. One test is rated 

and the others are rated with an R.
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Quality Control The following are recommended minimum procedures for quality 

control purposes:

◆ Have a different reader do a second examination of every 5th 

negative slide for beginners. Experienced readers should have 

every 10th negative slide re-read.

◆ Include slides of known negative and positive controls to test 

readers and verifiers.

Training Technicians and Verifiers

Background To assure accuracy and efficiency when laboratory personnel are 

processing samples, technicians need to be appropriately trained and 

successfully complete documented procedures. Technicians should 

complete the training program before actually processing real 

samples.

Bunted kernel verifiers should receive appropriate training, including 

identification and equipment usage. Spore verifiers, as well, should 

have training in examining isolates of Tilletia indica, and should meet 

certain educational and experience requirements. Slide readers should 

receive all laboratory procedure training, including gross teliospore 

identification and slide preparation. Written training records for all 

analysts covering all procedures will be maintained at the laboratory.

Suggested training content for all laboratory personnel

◆ Presenting general orientation about Karnal bunt

◆ Identifying and explaining the purpose of processing areas

❖ Setting up the reception (sample receiving) area

❖ Setting up the dirty rooms

❖ Setting up the clean rooms

◆ Receiving samples

❖ Maintaining supplies

❖ Decontaminating samples

❖ Logging-in samples

❖ Storing reserve samples

Important

Do not allow technicians involved in bunted kernel analysis or field personnel 

to enter the laboratory areas where seed analysis is taking place.
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◆ Extracting samples

❖ Maintaining supplies

❖ Preparing Shear’s mounting fluid

❖ Preparing sample containers

❖ Weighing and measuring samples

❖ Adding extraction solution

❖ Extracting the samples

❖ Cleaning up the extraction lab

◆ Preparing slides

❖ Maintaining supplies

❖ Setting up the working area

❖ Establishing priority and processing sample tubes

❖ Preparing the slides

❖ Making labels and data sheets

❖ Handling spills and cleanup

◆ Examining slides for Karnal bunt

❖ Selecting and examining slides

❖ Identifying Karnal bunt teliospores

❖ Verifying teliospores (for spore verifiers)

❖ Counting teliospores, if appropriate

❖ Reading positive slides

❖ Keeping records and interpreting laboratory results

◆ Examining wheat kernels for Karnal bunt (automated and 

manual methods)

❖ Maintaining supplies

❖ Processing samples

❖ Preparing the workstations

❖ Identifying Tilletia indica from bunted kernels

❖ Storing samples

❖ Decontaminating equipment and cleaning up
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Selecting and Training Bunted Kernel Verifiers

Background The Karnal bunt bunted kernel verifier must be able to positively 

identify Tilletia indica Mitra, the causal agent of Karnal bunt, in or on 

the wheat grain kernel to the exclusion of other fungi. The bunted 

kernel verifier serves as the final authority for the identification of 

Karnal bunt from grain samples.

Requirements The bunted kernel verifier should have work experience in microscopy, 

or other work experience in an agricultural or biological field. The 

verifier must pass the bunted kernel identification course given by a 

PPQ Identifier.

Job Description The incumbent is responsible for verifying the identification of 

T. indica bunted kernels found during Karnal bunt screening to the 

exclusion of other fungi. This identification will be based on a visual 

examination of infected grain. Symptoms must be verified with a 

microscopic examination of Karnal bunt teliospore gross 

morphological characteristics.

Training Kernel verifiers need to complete the following training activities:

◆ Receive introduction and background information on Karnal 

bunt and training and testing from a PPQ Plant Pathology 

Identifier.

◆ Learn about Tilletia indica, specifically:

❖ Life cycle

❖ Initial infection

❖ Morphology of symptoms at various stages of infection

❖ Teliospore morphology

◆ Recognize other fungi that infect wheat kernels:

❖ Black point symptoms (Helminthosporium sativum, 

Alternaria sp.)

❖ Common bunt (T. laevis, T. tritici) kernel infection/teliospore 

morphology

❖ Dwarf bunt (T. controversa) kernel infection/teliospore 

morphology

◆ Learn slide making and record keeping procedures.

◆ Observe grain examination machine demonstration and practice 

runs with known infected samples.

◆ Examine specimens.
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Identification 

Authority

After the incumbent completes the required training and shows the 

ability, through testing or correct identifications, to identify T. indica

bunted kernels in Karnal Bunt Project samples, designated USDA/

APHIS personnel (PPQ Plant Pathology Identifiers) will grant 

identification authority for use on the Karnal Bunt Project.

Selecting and 

Training Spore 

Verifiers

The Karnal bunt teliospore verifier must be able to positively identify 

Tilletia indica Mitra, the causal agent of Karnal bunt, to the exclusion 

of other fungi. The verifier serves as the final authority for identification 

of Karnal bunt.

Requirements

The verifier should hold a college degree in mycology, plant pathology, 

or other related fields. The verifier should have work experience with 

microscopy, fungal taxonomy, and morphology, or course work in plant 

pathology and mycology and work related experience in plant pathology 

and mycology. Knowledge or experience in plant pathology is desirable 

but not a requirement. For those without a degree in plant pathology or 

a related field, the requirement should be a foundation in mycology or 

plant pathology with recent work experience in that area (for example, 

PPQ Port Identifier or backup, in plant pathology).

Job Description (the incumbent is responsible for these duties)

 1. Verifies the identification of T. indica teliospores found by 

microscopists during Karnal bunt screening to the exclusion of 

other fungi.

 2. Distinguishes by microscopic examination other species of 

Tilletia that could be encountered (for example, T. walkeri and 

T. horrida).

 3. Using established guidelines on spore size, makes 

determinations of when to send “suspect spores” to National 

Mycologist for final determination.

 4. Confirms wheat kernels infected by T. indica.

Training

Training will consist of microscopic examination of several isolates of T. 

indica. Depending on their age and condition, teliospores will exhibit 

significant variation in size, shape, and color. Verifiers will frequently 

find immature and fragmented spores.

Karnal bunt teliospore verifiers will complete the following training 

activities:

◆ Analyze slides of T. indica, T. horrida (rice smut), and T. walkeri.

◆ Review microscope slides of actual samples to familiarize 

themselves with the other fungi commonly found associated with 

wheat and pollen grains frequently encountered.
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◆ Scan a minimum of 10 training slides of actual wheat samples, 

with known numbers of teliospores. At least one of these slides will 

be a negative control. The purpose of this training slide exercise is 

to familiarize the verifiers with the microscopist’s tasks, not for 

them to become skilled microscopists. For the verifier to qualify as 

a reader, the verifier must meet the requirements for readers 

described in the previous subsection, Training Technicians.

◆ Examine wheat kernels macroscopically and learn how to 

positively confirm a bunted kernel caused by T. indica.

Retesting

A system of retesting verifiers should be in place. This should include 

examining slides with various wheat smuts, particularly variations of 

Tilletia species.

Identification Authority

After the candidate completes the required training and shows the 

ability to identify T. indica teliospores and bunted kernels in Karnal 

Bunt Project samples, designated USDA personnel (PPQ National 

Mycologist or designee) will grant identification authority for use on the 

Karnal Bunt Project.

Preparing New Sieves for Use

Background New sieves generally arrive at the laboratory by mail from the USDA 

manufacturer. These sieves should arrive pre-labeled with the mesh 

size (20 or 53μ), month and year of manufacture, and unique serial 

number. Each size should be uniquely color coded. Upon receipt of 

newly manufactured sieves, it is the laboratory’s responsibility to 

ensure the quality of each sieve prior to use.

Step 1—Log the date received.

For each new sieve, log the date received at the laboratory and that 

sieve’s unique serial number into the Karnal Bunt Sieve Log.

Step 2—Examine the sieve.

Using a microscope equipped with an ocular micrometer, verify the 

quality and mesh size of each sieve.

◆ Examine the entire mesh under the microscope to look for tears, 

holes, or gaps in the mesh (recommended at 40x).

Important

Because sieves have a limited life, quality control measures require 

replacement at prescribed intervals. It is important to anticipate future needs 

for sieve replacement well in advance of when new sieves are required.
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◆ Measure the mesh pore size with an ocular micrometer at any 

location where a gap in the mesh appears, and additionally at 

five or more separate random locations across the mesh 

(recommended at 200x).

◆ Examine the silicone seals. If the silicone seal is imperfect and 

can be repaired, touch up with silicone; otherwise it is unusable. 

After a silicone application, do not place the sieve into service for 

24 hours.

If you find no damage, the mesh size is correct, and the silicone seal is 

intact, the sieve can go into service following step 3 below. If the sieve 

is unusable due to damage, improper mesh size, or an unsalvageable 

silicone seal, the sieve cannot be used, and must be destroyed. The 

person performing the verification enters the results into the Karnal 

Bunt Sieve Log. When a sieve is destroyed, log the date and your 

initials into the Karnal Bunt Sieve Log sheet.

Step 3—Place the sieve into use.

Each sieve that passes the examination can be placed into use. Log 

the date into use on the Karnal Bunt Sieve Log. Then soak the sieve in 

soapy water for 15 minutes and rinse with clear water. After rinsing, 

soak the sieve in 30 percent bleach solution for an additional15 

minutes and rinse once more in clear water. Log the serial numbers of 

the sieves that are now ready for use into the spaces across the top 

row of the Karnal Bunt Sieve Usage Log. These sieves can now be used 

for sample extraction.

Maintaining Sieves

Background To ensure that intact sieves are used to extract samples, inspect the 

sieves before each use.

Step 1—Record the sieve’s use.

Record every use of each sieve into the Karnal Bunt Sieve Usage Log. 

In the column below each sieve’s serial number on the log sheet, enter 

the sample/sub-sample number that the sieve is used to extract. 

Before performing an extraction, visually inspect every sieve to be 

used for any tear in the mesh or other imperfection, such as damaged 

silicone. If you find a sieve to be unusable, destroy it. Record the date, 

your initials, and the reason destroyed on Karnal Bunt Sieve Log 

sheet. Also record your initials and the date destroyed at the bottom of 

the column for that sieve’s serial number on the Karnal Bunt Sieve 

Usage Log.
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Step 2—Recheck the sieve.

After ten uses, recheck each sieve for quality as in Step 2 above. 

Record your initials and the date of damage check on the Karnal Bunt 

Sieve Usage Log in the space below the 10th extraction set entry. If 

you find the sieve to be usable, soak it in warm soapy water for 15 

minutes and rinse in clear water before further use. If you destroy the 

sieve at this point, record the action in both the Karnal Bunt Sieve Log 

and Sieve Usage Log. Perform these rechecks after the tenth and 

twentieth uses. Take each sieve out of service and destroy it after 

30 uses, regardless of the sieve’s condition at that point.

Ordering Sieves

Sieves are manufactured at the USDA, APHIS, PPQ Aircraft and 

Equipment Operations Center in Mission, Texas. If you need 

replacement sieves, fax a request including the number of sets 

required, the requested date of delivery, and delivery information.

Contact the Operations Center at the following numbers:

◆ Fax: (956) 580-7276

◆ Phone: (956) 580-7270

Forms

Each laboratory location will develop forms used for recording results 

of bunted kernel and teliospore analyses. Assure that the appropriate 

levels of information collection are included on each data collection 

form. Refer to samples of such forms on the following pages.
Appendix D Laboratory Procedures

Forms

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FIGURE D-3: Karnal Bunt Kernel Analysis Worksheet
Appendix D Laboratory Procedures

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FIGURE D-4: Karnal Bunt Quality Control (QC) Worksheet for Bunted Kernels
Appendix D Laboratory Procedures

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FIGURE D-5: Quality Control (QC) Grain Labs
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FIGURE D-6: Tilletia indica Spore Analysis Worksheet
Appendix D Laboratory Procedures

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FIGURE D-7: Tilletia indica Microscopy Log Sheet